靶向siRNA^CD31_脂质体复合物对人肺微血管内皮细胞PECAM-1表达的影响

Effect of targeted siRNA^CD31-lipofectamin complex on PECAM-1 expression in human pulmonary microvascular endothelial cells

目的研究以血管内皮细胞为靶向的siRNA^CD31’对血小板内皮黏附分子1（PECAM-1）表达及细胞增殖的影响。方法实验分为裸siRNA^CD31组、siRNA^CD31FAM组、稳定阴性对照（SNC）组、空白对照组。实验以阳离子脂质体（RNAi-mate）为载体，将2’-O-甲基修饰的siRNA^CD31转染体外培养的人肺微血管内皮细胞（HPMVEC），四甲基偶氮唑蓝（MTT）法检测siRNA^CD31对HPMVEC增殖的影响，RT-PCR、Westernblot检测PECAM1的表达水平。结果裸siRNA^CD31和siRNA^CD31FAM转染的HPMVEC的PECAM-1mRNA、PECAM-1蛋白的表达量、HPMVEC的增殖率均低于SNC组和空白对照组（P值均〈0．01），裸siRNA^CD31和siRNA^CD31-FAM转染的HPMVEC的PECAM1mRNA、PECAM-1蛋白的表达量、HPMVEC的增殖率差异无统计学意义（P值均〉0．05）。结论靶向siRNA^CD31可下调HPMVEC的PECAM-1mRNA和蛋白的表达，并可抑制HPMVEC细胞的增殖，2’O甲基修饰的siRNA^CD31可作为沉默靶基因PECAM-1的有效的技术手段，RNAimate可作为siRNA^CD31的转染靶基因的载体，PECAM1可能是干预肺癌癌细胞微环境中的微血管内皮细胞功能的潜在靶标。

Objective To investigate the effect of siRNA^CD31 targeting to vascular endothelial cells on platelet endothelial cell adhesion molecule 1 （PECAM 1） expression and endothelial cell proliferation. Methods Four groups were treated respectively by naked siRNA^CD31, siRNA^CD31FAM, stable negative control （SNC） and Opti-MEM （the blank control）. The chemically synthesized 2＇-O-methyl modification of siRNA^CD31 was transfected into human pulmonary microvascular endothelial cells （HPMVEC） using cationic lipofectamin complexes （RNAPmate）. The proliferation of HPMVEC was detected by MTT assay. The expression levels of PECAM-1 mRNA and protein were determined by semi-quantitative real-time PCR and Western blot respectively. Results The expression of PECAM1 mRNA and protein in HPMVEC and the proliferation of HPMVEC in naked siRNA^CD31 group and siRNA^CD31 FAM group were lower than those in SNC group and blank control group （all P 〈0.01）. Those results in naked siRNA^CD31 group and siRNA^CD31-FAM group were not different statistically （all P 〈0.05）. Conclusions Targeted 2＇ O-methyl-siRNA^CD31 may downregulate the expression of PECAM-1 mRNA and protein in HPMVEC and inhibit the proliferation of HPMVEC,which indicates 2＇-O-methyl-siRNA^CD31 can execute effective silence of the targeting gene PECAM 1 in HPMVEC. RNAi-mate may be used as a transfection vehicle of 2r-O methyl- siRNA^CD31. PECAM-1 may be a potential target on microvascular endothelial cells in tumor microenvironment.